Methylation of the N-terminal amino acid for many proteins has been observed, including when the N-terminal amino acid is methionine, alanine, phenylalanine and proline (Stock et al., (1987) FEBS Letters 220:8-14). In pilins expressed in Pseudomonas aeruginosa, the methylation of a number of different N-terminal amino acids has been postulated to be dependent on a fifth position glutamate (Pasloske and Paranchych, (1988) Molecular Microbiol. 2(4):489-495). Amino acids that were methylated at the N-terminus were alanine, glycine, tyrosine and methionine, while serine and phenylalanine were not (Strom and Lory, (1991) J. Biol. Chem. 266:1656-1664).
Recently, several signal transduction proteins have been shown to be methylated at glutamic acid side chains and/or at C-terminal amino acids (Stock and Lukat, (1991) Ann. Rev. Biophys. Chem. 20:109-136; Morgan et al., (1993) J. Bacteriol. 175:133-140). In bacteria, the activities of signal transducing proteins are regulated by methylesterification at glutamic acid side chains. Methyl accepting chemotaxis proteins are immunogenic and demonstrate high antigenic relatedness (Morgan et al., (1993) J. Bacteriol. 175:133-140). Antibodies against one methylated protein can crossreact with a very distinct spectrum of other methylated proteins with sequence identities of only 60 percent (Morgan et al., (1993) J. Bacteriol. 175:133-140). Paik, (1984) Methods Enzymol. 106:265-268, has suggested that methylation of the N-terminal amino acid may affect the physiological characteristics of proteins.
Recently, N-monomethylmethionine was observed at the N-termini of the ribosomal protein L16 (Brosius and Chen, (1976) FEBS Letters 62:105-109) and the bacterial chemotaxis protein CheZ (Stock et al., (1987) J. Biol. Chem. 262:8011-8014; Stock and Stock, (1987) J. Bacteriol. 169:3301-3311). There is some sequence similarity between these two proteins at the N-terminal end: EQU L16 (E. coli) N-methyl-Met-Leu-Gln-Pro- (SEQ. ID NO. 1) EQU CheZ (E. coli) N-methyl-Met-Met-Gln-Pro- (SEQ. ID NO. 2)
Stack, (1987) Advances in Post-translational Modification of Proteins and Aging, edited by Zappia, pp. 387-399, has suggested that the N-terminal sequence in CheZ and L16 is a signal of methylation for N-terminal methionine. The level of methylation reported in the literature is in the range of 30-50 percent. The mechanism of methylation is unknown, although it is known that it is dependent on S-adenosylmethionine. Because of CheZ, the process of methylation was linked with chemotaxis, but experiments indicated that no chemotaxis proteins are involved in methylation of CheZ. CheZ was expressed from a plasmid vector with the CheZ gene inserted behind the lac promoter in E. coli (JM109) that produces no flagellar or chemotaxis proteins. The first cycle of Edman degradation of this protein produced equal amounts of pth-Met and pth- N-methylmethionine (Stock et al., (1987) J. Biol. Chem. 262:8011-8014). Thus, CheZ methylation does not appear to be catalyzed by any other chemotaxis protein. It has been suggested that CheZ is self methylated or there is another unidentified methyl-transferase different than glutamate methyl-transferase involved in methylation of methionine. In addition, IF-3 is an E. coli protein that presents an N-terminal methylmethionine, but it has a glycine in position 4 (Brauer et al., (1977) FEBS Lett. 79:269-275).
There are at least ten other sequences of L16 which have been reported recently, but unfortunately all of them are translations of nucleic acid sequences (Atlas of Proteins and Genomic Sequences (1992) National Research Foundation, Compact Disc Edition). In most of them, Gln in the position 3 is mutated to Ser, which makes the sequences identical with the sequence of the dialpha chain of a recombinant hemoglobin known as rHb1.1 (Hoffman et al., WO 90/13645, published Nov. 15, 1990). EQU L16 sequences Met-Leu-Ser-Pro- (SEQ. ID NO. 3) EQU rHb1.1 (dialpha chain) Met-Leu-Ser-Pro- (SEQ. ID NO. 4)
Because these protein sequences are translations of gene sequences, there has not been any information about methionine methylation until now. DiDonato et al., (1983) J. Biol. Chem. 258:11890-11895, have achieved carboxymethylation of alpha amino groups of hemogobin using chemical means (reductive alkylation). Brunner et al., achieved acetylation of heterologous peptides by using a growth media low in amino acids (PCT Application WO 90/10706, published Sep. 20, 1990).
Besides the extent of methylation of an N-terminal amino acid, the extent to which the N-teminal methionine is processed (removed) has been postulated to be affected by the amino acid in position 2 (Hirel, et al., (1989) Proc. Natl. Acad. Sci USA 86:8247-8251). It was shown that glycine, alanine, proline, serine, threonine and cysteine appear to initiate N-terminal methionine processing, while Fujiyama and Tamanoi, (1990) J. Biol. Chem. 265:3362-3368 showed that RAS2, a S. cerevisiae protein undergoes N-terminal methionine removal with a proline in position 2.